Eukaryotic organisms employ a suite of epigenetic regulatory systems that are essential for genome stability and normal gene expression. Disruptions in epigenetic systems are associated with a wide range of pathologies, including chromosome instability, birth defects, cancer and developmental abnormalities. An intriguing type of epigenetic regulation, X chromosome dosage compensation, adjusts the expression of X-linked genes in one sex to accommodate the different numbers of X chromosomes in males and females. Dosage compensation has been intensively studied in mice, C. elegans and Drosophila. The striking differences in the dosage compensation strategies of these organisms are usually emphasized. However, all three systems rely on chromatin regulatory complexes that are selectively recruited to the X chromosome to modulate expression. These complexes, and their actions on chromatin, have been studied in great detail. But in no case do we understand how X chromatin is identified with the requisite selectivity. Our long-term goal is to understand how an entire chromosome is identified and regulated. The specific hypothesis we will test is that X chromosome recognition in Drosophila involves small RNAs. Our studies reveal that bi-directionally transcribed satellite sequences that are near-exclusive to the X chromosome respond to a potent genetic modifier of compensation. We postulate that the genetic modifier exerts its effect through siRNA. The proposed experiments will determine the role of small RNA pathways and X-linked satellites in dosage compensation. Three specific aims will be pursued to determine if 1) RNAi normally influences X chromatin and dosage compensation, 2) if X-linked satellites are sources and/or targets of RNAi, and 3) if X- linked satellites contribute to the dosage compensation of genes situated nearby. We propose that RNAi acts prior to, or in parallel with previously identified elements, which are modestly enriched on the X chromosome, to ensure exclusive recognition of X chromatin. PUBLIC HEALTH RELEVANCE: Proper regulation of the genome requires coordinated control of groups of genes as large as an entire chromosome. This proposal will investigate the idea that small non- coding RNA (RNAi), acting on repetitive sequences that are limited to particular chromosomes, is an intrinsic feature this process in flies. The similarities between analogous processes in flies and mammals suggest that this mechanism may also contribute to normal genome regulation in humans.